From the Departments of Anesthesiology and Pharmacology (A.F.Y.C., M.T., H.K., Z.S.K.), Divisions of Endocrinology and Metabolism (T.O.), Cardiovascular Diseases (V.J.P.), and Anatomic Pathology (T.B.C.), Mayo Clinic, Rochester, Minn, and the Department of Microbiology and Immunology (D.J.S.), Pennsylvania State University College of Medicine, Hershey.

From the Departments of Anesthesiology and Pharmacology (A.F.Y.C., M.T., H.K., Z.S.K.), Divisions of Endocrinology and Metabolism (T.O.), Cardiovascular Diseases (V.J.P.), and Anatomic Pathology (T.B.C.), Mayo Clinic, Rochester, Minn, and the Department of Microbiology and Immunology (D.J.S.), Pennsylvania State University College of Medicine, Hershey.

From the Departments of Anesthesiology and Pharmacology (A.F.Y.C., M.T., H.K., Z.S.K.), Divisions of Endocrinology and Metabolism (T.O.), Cardiovascular Diseases (V.J.P.), and Anatomic Pathology (T.B.C.), Mayo Clinic, Rochester, Minn, and the Department of Microbiology and Immunology (D.J.S.), Pennsylvania State University College of Medicine, Hershey.

From the Departments of Anesthesiology and Pharmacology (A.F.Y.C., M.T., H.K., Z.S.K.), Divisions of Endocrinology and Metabolism (T.O.), Cardiovascular Diseases (V.J.P.), and Anatomic Pathology (T.B.C.), Mayo Clinic, Rochester, Minn, and the Department of Microbiology and Immunology (D.J.S.), Pennsylvania State University College of Medicine, Hershey.

From the Departments of Anesthesiology and Pharmacology (A.F.Y.C., M.T., H.K., Z.S.K.), Divisions of Endocrinology and Metabolism (T.O.), Cardiovascular Diseases (V.J.P.), and Anatomic Pathology (T.B.C.), Mayo Clinic, Rochester, Minn, and the Department of Microbiology and Immunology (D.J.S.), Pennsylvania State University College of Medicine, Hershey.

From the Departments of Anesthesiology and Pharmacology (A.F.Y.C., M.T., H.K., Z.S.K.), Divisions of Endocrinology and Metabolism (T.O.), Cardiovascular Diseases (V.J.P.), and Anatomic Pathology (T.B.C.), Mayo Clinic, Rochester, Minn, and the Department of Microbiology and Immunology (D.J.S.), Pennsylvania State University College of Medicine, Hershey.

From the Departments of Anesthesiology and Pharmacology (A.F.Y.C., M.T., H.K., Z.S.K.), Divisions of Endocrinology and Metabolism (T.O.), Cardiovascular Diseases (V.J.P.), and Anatomic Pathology (T.B.C.), Mayo Clinic, Rochester, Minn, and the Department of Microbiology and Immunology (D.J.S.), Pennsylvania State University College of Medicine, Hershey.

From the Departments of Anesthesiology and Pharmacology (A.F.Y.C., M.T., H.K., Z.S.K.), Divisions of Endocrinology and Metabolism (T.O.), Cardiovascular Diseases (V.J.P.), and Anatomic Pathology (T.B.C.), Mayo Clinic, Rochester, Minn, and the Department of Microbiology and Immunology (D.J.S.), Pennsylvania State University College of Medicine, Hershey.

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Abstract

Endothelial NO synthase (eNOS) is an enzyme responsible for the production of a potent vasodilator and a key regulator of vascular tone, NO. In peripheral arteries, expression of a recombinant eNOS gene increases production of NO in the blood vessel wall. This approach appears to be a promising strategy for gene therapy of cerebrovascular disease. The major objective of the present study was to determine whether a recombinant eNOS gene (AdCMVNOS) can be functionally expressed in cerebral arteries. Replication-defective recombinant adenovirus vectors encoding bovine eNOS and Escherichia coli beta-galactosidase (AdCMVLacZ) genes, driven by the cytomegalovirus promoter, were used for ex vivo gene transfer. Rings of canine basilar artery were incubated with increasing titers of the vectors in MEM. Twenty-four or forty-eight hours after gene transfer, expression and function of AdCMVNOS were evaluated by (1) immunohistochemical staining, (2) isometric tension recording, and (3) cGMP radioimmunoassay. Transfection with AdCMVNOS resulted in the expression of recombinant eNOS protein in the vascular adventitia and endothelium, associated with significantly reduced contractile responses to UTP and enhanced endothelium-dependent relaxation to calcium ionophore A23187. Basal production of cGMP was significantly increased in the transfected vessels. The reduced contractions to UTP with increased cGMP production were reversed by a NOS inhibitor, NG-monomethyl-L-arginine. Contractions to UTP or production of cGMP were not affected in arteries transfected with AdCMVLacZ reporter gene. The results of the present study represent the first successful transfer and functional expression of recombinant eNOS gene in cerebral arteries. Our findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall. (Circ Res. 1997;80:327-335.)

Introduction

Nitric oxide plays an essential role in the regulation of cerebrovascular tone under both physiological and pathological conditions. [1–4] Three distinct genes encoding NOS isoforms have been cloned. [1] In cerebral arteries, the constitutive NOS in endothelial cells [5,6] and perivascular nerves [7,8] is responsible for NO production under normal conditions and in response to a variety of chemical and physical stimuli. [1–4] The inducible isoform of NOS, in contrast, produces much greater amounts of NO in smooth muscle cells in response to immunostimulants, such as endotoxin, tumor necrosis factor, and certain cytokines. [1–4] Cerebral arterial tone, therefore, may be modulated in a complex manner by changes in expression of constitutive and/or inducible NOS activity with subsequent alteration of local NO production. [2]

Adenovirus vectors have been used to achieve efficient transfer and expression of recombinant genes in different vasculatures both ex vivo and in vivo, raising the possibility that this approach may be used to treat vascular disorders. [9,10] The advantages of these vectors for transferring genes to the vascular system have recently been described. [10] Luminal administration of adenovirus vectors has been used to target genes to the endothelial layer. [11] Smooth muscle cell transfection is achieved when the vessel is injured at the time of transfection. [12,13] More recently, perivascular adenoviral transfection of the reporter gene encoding beta-galactosidase to the baboon carotid [14] and the rat cerebral blood vessels [15] has been described. Thus, it is possible to target gene transfer to a specific layer of the vessel wall by luminal or periadventitial administration of vector. The latter method is an attractive approach to transfection of cerebral blood vessels because of significant problems associated with the interruption of cerebral blood flow, which is required for luminal administration of recombinant DNA. More important, periadventitial administration of antisense oligonucleotides has been shown to inhibit neointimal formation in the injured rat carotid artery, [16] suggesting that this mode of gene transfer may result in demonstrable biological effects.

Von der Leyen et al [17] have reported that direct transfer of eNOS cDNA mediated by the Sendai virus (HVJ) in a DNA-nuclear protein-liposome complex prevents neointimal formation after balloon injury in the rat carotid artery. [17] More recently, it has been demonstrated that expression of human recombinant eNOS gene in rat lungs may prevent hypoxic vasoconstriction. [18] Furthermore, Tzeng et al [19] in 1996 successfully used a retrovirus vector to deliver the human recombinant inducible NOS gene to porcine femoral arteries. [19] However, eNOS gene transfer, functional expression, and effect on cerebrovascular tone have not been investigated. The present study was therefore undertaken to determine whether adenovirus-mediated transfer of the eNOS gene may affect the vascular tone of the canine basilar artery.

Materials and Methods

Construction, Propagation, and Purification of Adenovirus Vector

Recombinant adenovirus containing the cDNA encoding eNOS was generated (Figure 1) as described previously. [20] Briefly, the shuttle vector, pACCMVpLpA, was a kind gift of Dr Robert Gerard (University of Texas Southwestern Medical Center, Dallas, Tex). The plasmid containing cDNA for bovine aortic endothelial cell eNOS was generously provided by Dr David G. Harrison (Emory University, Atlanta, Ga). The eNOS cDNA was inserted into pACCMVpLpA. The resulting plasmid was linearized with Nru I and cotransfected with dl309 into 293 cells by calcium phosphate/DNA coprecipitation. dl309 is a biologically selected, restriction enzyme site-loss variant of wild-type adenovirus type 5, which retains only a single Xba I site at nucleotide 1339. [21] The 293 cells are human embryonic kidney carcinoma cells that have been transformed with the left end of human adenovirus type 5 DNA. [22] Recombinant adenovirus vectors were generated by homologous recombination. [20] Viral plaques were picked and propagated in 293 cells. Viral DNA was enriched by Hirt extraction [23] and screened by restriction mapping and polymerase chain reaction for the presence of eNOS cDNA. Positive plaques underwent two further rounds of plaque purification in 293 cells. Stocks were prepared from positive plaques, and these were used to generate high-titer preparations. Viral preparations were performed by infecting a confluent monolayer of 293 cells in T175 flasks with viral stock at an MOI of 1 to 10. Virus was purified by double cesium gradient ultracentrifugation and was dialyzed against 10 mmol/L Tris, 1.0 mmol/L MgCl2, 1.0 mmol/L HEPES, and 10% glycerol for 4 hours at 4 degrees C. Viral titer was determined by plaque assay. [20] eNOS activity was measured in cultured porcine coronary smooth muscle cells transfected for 48 hours with AdCMVNOS at an MOI of 200. The cells acquired eNOS enzymatic activity as quantified by measuring [sup 3 H]L-citrulline formation from [sup 3 H]L-arginine. [1] The activity of eNOS was almost abolished by EGTA (1 mmol/L), a calcium chelator, or L-NMMA (0.1 mmol/L), a NOS inhibitor (data not shown). The enzyme activity was also confirmed by positive NADPH diaphorase staining [24,25] in confluent 293 cells transfected with AdCMVNOS (data not shown). The defectiveness of AdCMVNOS for replication was tested by adding the virus (107 to 108 pfu/mL) to a monolayer of diploid human embryonic lung fibroblasts (60-mm dish). Replication-competent viruses, at an MOI of 10 or more, produced a cytopathic effect and destroyed the monolayer in <3 days. Infection with AdCMVNOS at a comparable MOI produced no observable cytopathic effect after 5 days. The sensitivity for detection of replication-competent virus was 107 to 108 pfu/mL. AdCMVLacZ, used in all experiments as a control, was a kind gift of Dr James M. Wilson (University of Pennsylvania, Philadelphia). It was propagated, isolated, and quantified as described above.

Gene Transfer

Experiments were performed ex vivo on rings (4 mm long) of basilar arteries taken from mongrel dogs (18 to 27 kg) anesthetized with 30 mg/kg sodium pentobarbital administered intravenously. The arteries were initially transfected with three different titers of adenovirus vectors diluted in MEM for 30 minutes at 37 degrees C and then transferred to fresh MEM and incubated for 24 or 48 hours at 37 degrees C in a CO sub 2 incubator (Forma Scientific, Inc). All procedures were performed in accordance with the institutional guidelines of the Mayo Clinic.

Histochemical and Immunohistochemical Analysis of Gene Expression

For histochemical staining of beta-galactosidase, the vessels were fixed for 30 minutes in 2% paraformaldehyde/0.2% glutaraldehyde in PBS. They were then rinsed with PBS and placed in X-Gal reagent for 4 hours. [26] The stained vessels were dehydrated through graded alcohol to xylene washes and embedded in paraffin. Serial 5-micro m sections were lightly counterstained with eosin. For immunohistochemical staining of recombinant eNOS, arterial rings were frozen in O.C.T. (Miles, Inc) compound, and serial 5-micro m sections were cut. After immersion fixation in acetone (4 degrees C) and 1% paraformaldehyde/EDTA, the sections were incubated in 0.1% sodium azide/0.3% hydrogen peroxide and then incubated with 5% goat serum/PBS-Tween 20 to block the nonspecific protein binding sites. An eNOS monoclonal antibody (5 micro g/mL, 1:50 of stock, Transduction Laboratory) was applied for 60 minutes at room temperature, followed by incubations with biotinylated rabbit anti-mouse F(ab prime)2 (1:300, 20 minutes) secondary antibody and peroxidase-conjugated streptavidin (1:300, 20 minutes) (Vector Laboratories, Inc). After a 30-second immersion in 0.1 mol/L sodium acetate buffer (pH 5.2), eNOS immunoreactivity was visualized with 3-amino-9-ethylcarbazole and hematoxylin counterstaining.

For control studies, the specificity of eNOS immunolabeling was examined by (1) omission of the primary eNOS antiserum in the incubation medium, (2) eNOS immunostaining of AdCMVLacZ-transfected vessels, and (3) immunostaining of AdCMVNOS-transfected vessels with an isotype-matched primary antibody of eNOS, a mouse monoclonal IgG1 against OPD4 (1:50 dilution, Dako).

Measurement of Vascular Reactivity

Twenty-four or 48 hours after beta-galactosidase and eNOS gene transfection, arterial rings were connected to isometric force-displacement transducers (Grass Instrument) and suspended in organ chambers filled with 25 mL of gassed (94% O2/6% CO2) Krebs-Ringer bicarbonate control solution (pH 7.4, 37 degrees C) consisting of (mmol/L) NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2 PO4 1.2, NaHCO3 25.0, EDTA 0.0026, and glucose 11.1. Isometric tension was recorded continuously. Gradual stretch of 1.0 g was applied to each ring segment until 3.0-g tension was reached, as determined by the contraction to UTP (10 micro mol/L). [5] After three washes with control solution, dose-response curves to different agonists were obtained with cumulative applications of each agonist. In some experiments, L-NMMA (0.1 mmol/L) was added to the organ chamber for 15 minutes before the agonist applications. Only one concentration-response curve was obtained in each tissue preparation. Contractions to UTP in the absence or in the presence of L-NMMA and relaxations to SNP or SIN-1 were obtained 48 hours after gene transfer and are expressed in grams. Endothelium-dependent relaxations to A23187 were studied 24 hours after gene transfer, since (1) endothelial cells were preserved after 24 but not 48 hours of incubation in MEM, and (2) UTP (10 micro mol/L)-elicited contractions were comparable between AdCMVLacZ- and AdCMVNOS-transfected vessels after 24 (see legend for Figure 5) but not 48 hours. To rule out the contribution of cyclooxygenase activity, concentration-response curves to A23187 were obtained in the presence of indomethacin (10 micro mol/L).

Measurement of cGMP

Twenty-four hours after gene transfer, L-arginine (0.1 mmol/L) or L-NMMA (0.1 mmol/L) was added to the incubation medium containing ring segments for 30 minutes to supplement the precursor of NO or to inhibit eNOS enzymatic activity, respectively. Indomethacin (10 micro mol/L) and 3-isobutyl-1-methylxanthine (1.0 mmol/L) were also added to eliminate the possible influences of endogenous cyclooxygenase and phosphodiesterase, respectively. The ring segments were then rapidly frozen in liquid nitrogen and subjected to cGMP measurement with the aid of a radioimmunoassay kit (Amersham) as previously described. [27]

To determine the effect of calcium in the production of cGMP, both an extracellular calcium chelator (EGTA, 1 mmol/L) and an intracellular calcium chelator (BAPTA-AM, 20 micro mol/L) were added to the calcium-free medium, and arteries were incubated for 60 minutes at 37 degrees C. This was followed by AdCMVNOS gene transfer experiment and 24-hour incubation without calcium.

Statistical Analyses

The concentration-response curves to UTP in control basilar arteries and in basilar arteries transfected with three different viral titers (Figure 4) were analyzed using RM-ANOVA. A total of 6, 12, and 6 dogs were used for gene transfer experiments in Figure 4A, Figure 4B, and Figure 4C respectively. Global effects of the different treatments were tested using RM-ANOVA. The post hoc pairwise contrasts between the control and treatment for each of the three gene transfer groups were made using Dunnett's two-tailed procedure to control type I error. Endothelium-dependent relaxations to A23187 in AdCMVNOS- and AdCMVLacZ-transfected vessels were compared using two-way ANOVA, with 6 dogs used for each group (Figure 5).

A, Concentration-response curves to UTP in control basilar arteries and basilar arteries transfected with increasing titers of AdCMVLacZ were studied 48 hours after gene transfer. Six dogs were used, with four rings obtained from each dog. One of the four rings was assigned to the control, and three rings were exposed to three increasing titers of AdCMVLacZ, respectively. No significant difference was found in increase in tension among control and gene-transfected groups (P=.4933 by RM-ANOVA). Tensions in each viral titer were also compared with the control value using Dunnett's procedure, and no significant difference was found (always P>.05). B, Concentration-response curves to UTP in control basilar arteries and basilar arteries transfected with increasing titers of AdCMVNOS were studied 48 hours after gene transfer. Twelve dogs were used, with four rings obtained from each dog. One of the four rings was assigned to the control, and three rings were exposed to three increasing titers of AdCMVNOS, respectively. A significant difference was found in control and AdCMVNOS of three viral titers (P<.001 by RM-ANOVA). A significant reduction in tension in AdCMVNOS-transfected vessels for each viral titer was found compared with the control value using Dunnett's procedure (always P<.05). C, Concentration-response curves to UTP in control basilar arteries and basilar arteries transfected with increasing titers of AdCMVNOS were studied 48 hours after gene transfer. Experiments were performed in the presence of L-NMMA. Six dogs were used, with four rings obtained from each dog. One of the four rings was assigned to the control, and three rings were exposed to three increasing titers of AdCMVNOS, respectively. No significant difference was found in increase in tension among control and gene-transfected groups (P=.5486 by RM-ANOVA). Tensions in each viral titer were also compared with the control value using Dunnett's procedure, and no significant difference was found (always P>.05).

To study the effects of gene transfer on the production of cGMP, totals of 7, 5, and 4 dogs were used for control, AdCMVLacZ, and AdCMVNOS, respectively (Figure 6A). The average of cGMP in LacZ- and in eNOS-treated vessels over the three viral titers was taken to compare with that of the control vessels. One-way ANOVA was used to compare the global differences across the three groups of vessels (control, LacZ, and eNOS) on cGMP levels. The post hoc pairwise comparisons were then made among the control, AdCMVLacZ, and AdCMVNOS groups using Bonferroni's procedure to control type I error. To study the effects of eNOS alone and eNOS in the presence of L-NMMA (Figure 6B) or eNOS in the presence of calcium (Figure 6C) on cGMP production, totals of 7 and 6 dogs were used, respectively. For Figure 6B, one-way ANOVA with repeated measures was used to test the global difference in the production of cGMP among the control group and the two treatment groups, together with pairwise comparisons using Bonferroni's procedure. For Figure 6C, a two-factor design with repeated measures on both factors (control/eNOS and presence/absence of calcium) was used to test the global difference among the levels of these factors with pairwise comparisons using Bonferroni's procedure.

A, Bar graphs showing cGMP levels in control basilar arteries and in basilar arteries transfected by increasing titers of AdCMVLacZ and AdCMVNOS. Seven dogs were used for control. Five dogs were used for AdCMVLacZ with repeated measures for three viral titers, and 4 dogs were used for AdCMVNOS with repeated measures for three viral titers. The average of cGMP levels in the AdCMVLacZ and AdCMVNOS groups over three viral titers was taken, and one-way ANOVA was used to compare the cGMP levels in the three treatment groups. Overall, there was a significant difference in production of cGMP among these three groups (P<.001). Pairwise comparisons with Bonferroni's adjustment also indicate that the production of cGMP in AdCMVNOS-transfected vessels was significantly higher than that of the control or that of the AdCMVLacZ-transfected vessels (always P<.05). B, Bar graphs showing basal cGMP levels in control basilar arteries and in basilar arteries transfected by AdCMVNOS and by AdCMVNOS in the presence of L-NMMA. Seven dogs were used, with three rings obtained from each dog. One of the three rings was assigned to the control group, and the remaining two rings were subjected to eNOS or eNOS+L-NMMA treatment. Overall, there was a significant difference in cGMP production among the three treatment conditions (P<.01 by RM-ANOVA). Pairwise comparisons with Bonferroni's adjustment also indicate that the cGMP production in AdCMVNOS-transfected vessels was significantly higher than that of the control or that of eNOS+L-NMMA-treated vessels (always P<.05). C, Bar graphs showing basal cGMP levels in control and in basilar arteries transfected by AdCMVNOS in the presence and in the absence of calcium. Six dogs were used, with four rings obtained from each dog. Two of the four rings were assigned to the control groups in the presence or absence of calcium, and the other two rings were subjected to eNOS treatment groups with or without calcium. A two-way RM-ANOVA was used, and a significant overall difference was detected (P<.005). Pairwise comparison with Bonferroni's adjustment also indicates that cGMP production in AdCMVNOS-transfected vessels in the presence of calcium was significantly higher than that of the controls and of eNOS in the absence of calcium (always P<.05).

The statistical analyses were carried out using the General Linear Models procedure of the Statistical Analysis System package (version 6.11, SAS Institute Inc). In all the tests, a two-tailed value of P<.05 was taken as evidence of a statistically significant finding.

Results

Expression of beta-Galactosidase Reporter Gene and eNOS Gene

Twenty-four or 48 hours after AdCMVLacZ transfection, recombinant beta-galactosidase protein was expressed in all canine basilar arteries studied (Figure 2). Recombinant eNOS protein was also expressed in arteries transfected with AdCMVNOS for either 24 hours (Figure 3A) or 48 hours (data not shown). Transfection of different vessel segments taken from the same animal resulted in a viral titer-dependent (109, 1010, and 1011 pfu/mL for AdCMVLacZ; 109, 1010, and 3.3x1010 pfu/mL for AdCMVNOS) increase in beta-galactosidase (Figure 2) and eNOS (data not shown) expression, respectively. Expression of both proteins was localized predominantly in the adventitia (Figure 2B and Figure 2C and Figure 3A and Figure 3B), whereas endothelium expression of recombinant proteins was also seen in the vessels incubated for 24 hours (Figure 2B, Figure 3A, and Figure 3B). Forty-eight hours after gene transfer, however, endothelial cells were not preserved in either the control (data not shown) or adenovirus vector-exposed arteries (Figure 2C). All nontransfected vessels lacked expression of both recombinant beta-galactosidase (Figure 2A) and eNOS (data not shown). In addition, AdCMVLacZ-transfected vessels did not show positive eNOS immunoreactivity (Figure 3D), and AdCMVNOS-transfected vessels stained negatively when an isotype-matched primary antibody of eNOS was used (ie, monoclonal mouse IgG1 against OPD4) (Figure 3E) and failed to demonstrate beta-galactosidase activity (data not shown).

Effects of Gene Transfection on Vascular Reactivity

In AdCMVLacZ-transfected vessel segments, relaxation responses to the NO-releasing agents SNP (0.1 mmol/L) and SIN-1 (0.3 mmol/L) were not significantly different compared with the response of the control vessels (Table 1). Similarly, the receptor-mediated contractile response to UTP (0.1 micro mol to 1.0 mmol/L) in AdCMVLacZ-transfected vessels was similar to that of the nontransfected control vessels (Figure 4A). The EC50 and maximal contractile responses to UTP in arteries transfected with AdCMVLacZ were also similar to the responses of the control vessel segments at all three viral titers (data not shown). In contrast, the contractile effect of UTP was significantly reduced in arteries transfected with AdCMVNOS compared with that of the control vessels (Figure 4B), and the reduction in contraction was significantly inhibited by L-NMMA (0.1 mmol/L, Figure 4C). In addition, endothelium-dependent relaxations to A23187 (1 nmol to 1 micro mol/L) in AdCMVNOS-transfected arteries were significantly enhanced compared with relaxations in AdCMVLacZ-transfected vessels (1010 pfu/mL, 24 hours after gene transfer, Figure 5).

Effects of Gene Transfection on Intracellular cGMP Levels

In basilar arteries transfected with AdCMVNOS at all three titers, the basal cGMP production was significantly elevated compared with cGMP levels in arteries transfected with AdCMVLacZ or nontransfected control vessels (Figure 6A). The cGMP levels in AdCMVLacZ-transfected vessels, on the other hand, were not significantly different when compared with the levels in control vessels (Figure 6A). The increased intracellular cGMP content in AdCMVNOS-transfected vessels was significantly reduced by the NOS inhibitor L-NMMA (0.1 mmol/L) (Figure 6B). In addition, the elevation of cGMP in AdCMVNOS-transfected vessels was abolished in the calcium-free medium in the presence of calcium chelators EGTA (1 mmol/L) and BAPTA-AM (20 micro mol/L) (Figure 6C).

Discussion

This is the first study to demonstrate the successful transfer and functional expression of a recombinant eNOS gene in large cerebral arteries, resulting in increased basal production of cGMP with a subsequent reduction in receptor-mediated contractile response and enhancement of endothelium-dependent relaxations. Our results indicate that adenovirus-mediated transfer and expression of the recombinant eNOS gene increases the apparent activity of NO in the arterial wall.

Adenovirus vector transfection of canine basilar arteries resulted in both beta-galactosidase reporter gene and eNOS gene expression. This was demonstrated by positive histochemical and immunohistochemical stainings of respective recombinant proteins in the arterial wall. The level of expression was dependent on viral titer.

To examine the effect of adenoviral transfection on vascular reactivity, responses of canine basilar artery to several known contractile and relaxing agents were analyzed using isometric tension recording. UTP, an endogenous pyrimidine nucleotide normally present in platelets and linked to storage organelles, has been shown to be a potent receptor-mediated vasoconstrictor of canine and human cerebral arteries. [28-30] In contrast, inorganic nitroso compounds such as SNP and sydnonimines such as SIN-1 are nitrovasodilators, producing relaxation of cerebral arteries as NO donors by either releasing NO spontaneously (ie, SNP) or as a result of reaction with molecular oxygen (ie, SIN-1). [31] In the present study, both UTP-elicited contractions and SIN-1-induced and SNP-induced relaxations in beta-galactosidase reporter gene-transfected basilar arteries were similar to those of the control vessels. These data strongly suggest that even with high titers of adenovirus vectors, short-term exposure (ie, 30 minutes) does not result in impaired vasoreactivity in canine basilar arteries ex vivo.

Although the experiments in the present study were performed ex vivo, it was considered important to find out the possible adverse effect of adenoviral transfection on cerebral vasomotor function. Several shortcomings associated with the first-generation adenovirus vectors include the relatively short duration of transgene expression, the host immune response to the viral proteins, and the possibility of becoming replication competent as a result of the functional similarities of some host cell proteins to the deleted E1a region of the virus. [10] Indeed, adenovirus-mediated gene transfer to the vessel wall has recently been shown to cause unwanted effects, including vascular cell activation, inflammation, and neointimal hyperplasia. [32] In addition, alteration of rabbit carotid arterial tone by ex vivo gene transfer with a replication-deficient adenovirus has been described in a preliminary report. [33] These findings may be explained by the fact that adenovirus vectors, particularly at high MOIs, are able to replicate, although more slowly. [22] In the present study, we found no evidence of vasomotor dysfunction even when relatively high viral titers were used (ie, up to 1011 pfu/mL). However, the possibility of adenoviral toxicity leading to vasomotor dysfunction in vivo needs to be investigated.

Extensive eNOS immunoreactivity localized in the adventitial layer of AdCMVNOS-transfected canine basilar arteries indicates that recombinant eNOS protein was expressed. However, successful gene transfer with subsequent protein expression does not necessarily result in a functional effect. Therefore, it was considered critically important to confirm whether expression of recombinant eNOS protein in the cerebral arteries resulted in corresponding changes in vasomotor activity. The results of the present study have demonstrated that endothelium-dependent relaxations to calcium ionophore A23187 are significantly enhanced in eNOS gene-transfected basilar arteries (ie, 1010 pfu/mL) studied 24 hours after exposure to AdCMVNOS. Moreover, 48 hours after gene transfer, contractions to UTP are significantly reduced. Since the contractile responses to UTP in beta-galactosidase reporter gene-transfected arteries of the same viral titers did not significantly differ from that of the control, it is logical to conclude that the effect of recombinant eNOS is due to the increased local production of NO rather than possible nonspecific effects of viral infection. The possibility of vascular smooth muscle cell damage due to the overexpression of recombinant eNOS should be minimal, since L-NMMA, an endogenous NOS inhibitor present in human plasma and urine, [34] reversed the suppressed contraction to UTP in AdCMVNOS-transfected vessels. These results are in agreement with conclusions of the studies by van der Leyen et al [17] and Janssens et al, [18] in which expression of a recombinant eNOS gene in rat carotid and pulmonary arteries reduced the contractile effects induced by KCl (50 mmol/L) or hypoxia, suggesting that expression of recombinant eNOS increases basal production of NO.

The viral titer-dependent suppression of contractions to UTP in AdCMVNOS-transfected vessels suggests an increased expression and activity of recombinant eNOS. This conclusion is further supported by the demonstration of increased basal cGMP production following eNOS gene transfer. Since NO exerts its effects through activation of soluble guanylate cyclase, an enzyme responsible for hydrolysis of GTP and formation of cGMP, [35,36] the concomitant increase in basal production of cGMP in eNOS gene-transfected arteries strongly supports the idea that expression of recombinant eNOS protein leads to enhanced NO generation. The specificity of the eNOS-elicited increase in cGMP production was confirmed by the observation that L-NMMA significantly reduced cGMP levels. Although the enzymatic activity of recombinant eNOS was not measured in the present study, functional expression of a recombinant eNOS gene has been demonstrated by immunohistochemical, pharmacological, and biochemical data.

Since the elevated cGMP production was calcium dependent, the involvement of inducible NOS activation by adenovirus vector is unlikely. This is consistent with our findings from functional studies demonstrating that endothelium-dependent relaxations induced by the calcium ionophore A23187 were significantly augmented in eNOS gene-transfected vessels compared with relaxations to A23187 obtained in reporter gene lacZ-transfected arteries. On the other hand, the tendency of cGMP increase in AdCMVLacZ-transfected arteries in increasing viral titers (up to 3.5x10 pfu/mL) did not result in any statistically significant difference compared with that of the nontransfected control arteries (Figure 6A), even though the possibility of nonspecific effects of viral protein and/or recombinant beta-galactosidase expressions on cGMP production cannot be excluded. These data are also consistent with the findings from functional studies, which demonstrated that in AdCMVLacZ-transfected arteries with increasing titers, the contractile responses to UTP were not reduced compared with responses of normal vessels.

Functional expression of eNOS gene with increased local NO production in the cerebrovasculature may have important clinical implications. Cerebral vasospasm that developed after subarachnoid hemorrhage, for instance, has been shown to be associated with an impaired L-arginine-NO-cGMP pathway, [6,37-41] even though there have been no reports so far on the possible genetic defects of the NOS gene family in vasospasm. Experimental vasospasm could be alleviated by intravenous administration of glycerol trinitrate, [42] a well-known nitrovasodilator that releases NO intracellularly, [31] intracarotid infusion of NO, [43] or restoration of endogenous NO production in the arterial wall by administration of L-arginine and superoxide dismutase. [44] Consistent with this notion, our approach was therefore to explore experimentally the possibility that expression of recombinant eNOS may result in "restored" NO activity, leading to improved vascular reactivity.

Perivascular expression of a recombinant gene (ie, AdCMVLacZ) can be achieved by infusing the adenovirus vectors into cerebrospinal fluid, as evidenced in experimental rats [14,15] and dogs (A.F.Y. Chen and Z.S. Katusic, unpublished data, 1996). The perivascular application of an adenovirus vector via cerebrospinal fluid with functional expression of recombinant eNOS in cerebral arteries may therefore become a potentially feasible therapeutic strategy in increasing local NO production, hence alleviating the vasospastic conditions. In addition, cerebral vasospasm is a diseased state that usually occurs between 4 and 12 days after the onset of subarachnoid hemorrhage. [45] Relatively short-term expression of the transgene (ie, 7 to 14 days in the vasculature) following transfection by adenovirus, [46] a basic problem of gene therapy in the treatment of chronic diseases, may be an advantage in gene therapy of cerebral vasospasm.

In summary, the present study demonstrates that eNOS gene can be functionally expressed in cerebral arteries, resulting in increased local NO production with subsequent increase in cGMP levels. We speculate that the approach of eNOS gene transfer may be useful in restoring the biosynthesis of NO in diseased blood vessels.

Acknowledgments

This study was supported in part by National Institutes of Health grants HL-44116 and HL-53542 (Dr Katusic), Mayo Clinic intramural research grants (Drs O'Brien and Pompili), and funds from the Mayo Foundation. Dr Alex F.Y. Chen was supported by National Institutes of Health training grant GM-08288 and the Mayo Clinic and Foundation. The authors would like to thank Dr Hongzhe Li (Mayo Clinic Biostatistics Division) for his valuable help in statistical analysis; Leslie Smith, Adele Stelter, Sharon Guy, and Steve Ziesmer for their technical assistance; and Janet Beckman for typing the manuscript.